R.T. Trout 1, C.D. Steelman 1, A.L. Szalanski 1, K. Kvamme 2, and P.C. Williamson 3 1 University of Arkansas Entomology Fayetteville, Arkansas University of Arkansas Anthropology Fayetteville, Arkansas University of North Texas Health Science Center Ft. Worth, Texas Genetic & Spatial Identification of Borrelia & Rickettsia Pathogens from Arkansas Ticks & Mammals

Tick - Host Associations Larvae Nymph Adult

Several Species Variable Symptoms Infection variable Cases –Misdiagnosed –Under reported Borrelia Pathogens Ixodes scapularisAmblyomma americanum B. burgdorferi B. lonestari

Spotted fevers Several Species Variable Symptoms –Fever, edema, spreading rash Infection variable Cases Dermacentor variabilisIxodes scapularis Amblyomma americanum Rickettsia Pathogens

Arkansas Tick-borne Diseases Changing State –Fragmented habitat –top-10 growing metropolitan areas (Anonymous 2001) 1996: military cases –R. rickettsii –R. typhus –B. burgdorferi (Warner et al. 1996)

Borrelia in Arkansas 26 cases of LD in Arkansas, since 2002

Rickettsia in Arkansas 231 cases of RMSF in 45 Ctys., since 2002

Objectives Genetically identify bacteria in Arkansas by examining ticks & hosts using molecular techniques –Borrelia pathogens –Rickettsia pathogens Identify potential risk areas using geographic information systems

Outline General Methods for the Project Results –Tick Collections –Borrelia from Ticks & Hosts –Rickettsia from Ticks –Co-infection Summary & Future Direction

Methods: Tick Collections Lyme disease risk increases in areas with Ab positive canines & ticks on white-tailed deer (Daniels et al. 1993)

Tick DNA- Ethanol Vials: Qiagen DNEasy Protocol Host DNA- Whatman FTA Cards: Protocol 4 Reactions 1 Tick DNA (16S mtDNA) 1 Borrelia (flaB) 2 Rickettsia (gltA & rompB) Methods: Extraction & PCR

Methods: PCR Tick DNA Reaction –16S mt DNA ribosomal RNA (Black & Piesman 1994) Borrelia genus specific reaction –flaB gene (Barbour et al. 1996) Rickettsia genus specific reactions –gltA (Kollars & Kengluecha 2001) –rompB (Regnery et al. 1991)

16S tick mtDNA Methods: Sample Verification Sequenced at UNT Health Science Center, Ft. Worth TX GenBank Map Incidence gltA rompB flaB

Methods: Host Distribution 247 Clinics 35 Participated (14%) Moran’s I: Deer (1.19) Pet (0.47)

Methods: GIS Mapping Results! IDW 2 Buffer Mask

Results: Tick Collections Canines –1498 ticks from 173 canines –8 ± 4.0 ticks/host (range 1-100) Felines –84 ticks from 16 felines –5 ± 2.4 ticks/host (range 1-17) Deer –1581 ticks from 250 deer –6 ± 0.3 ticks/host (range 0-32)

Results: Tick Collections

All ticks from pets were tested but Only 56% of the ticks collected from deer were tested

Ixodes scapularisAmblyomma americanum Results: Borrelia

Results: Borrelia in Ticks 57% Ticks from Canines Ticks from Deer 12% 24% 10% 36% 24% 43% 38% 33% 37% 23% 0% 27% 0% 34% 0% 34% 20% 42% Ticks from Felines

Results: Borrelia in Ticks Canines Deer Felines Total

Results: Borrelia species in ticks 613 ticks positive 3 B. burgdorferi 5 B. lonestari strains 22 SSc26 38 M2002-V1 21 Tx/06 44 Nc/Md 1 AA115 *487 ticks not sequenced

Results: Borrelia spp. in Hosts 15% 4% 25% Host Animal

Results: Borrelia in Hosts N = 1 N = 21 N = 3 N = 14

Results: Borrelia Incidence Ticks from Pets (n = 355 / 1585 ticks) Ticks from Deer n = 259 / 695 ticks)

Results: Borrelia Incidence Blood from Pets (n = 9/174 hosts) Blood from Deer (n = 55/220 hosts)

Results: Borrelia Incidence IDW 2 Ticks from Pets (n = 355 / 1585 ticks) Ticks from Deer n = 259 / 695 ticks)

Results: Borrelia Incidence IDW 2 Blood from Pets (n = 9/174 hosts) Blood from Deer (n = 55/220 hosts)

Results: Borrelia Incidence IDW 2 Map Algebra: (4 maps) * State Mask

Results: Borrelia Risk Areas 2007 Cases (n=8) Benton, Carroll, Crawford, Saline, Washington Co.

Results: Borrelia Risk Areas

Dermacentor variabilisIxodes scapularis Amblyomma americanum Results: Rickettsia

Results: Rickettsia in Ticks 33% Ticks from Canines Ticks from Deer 40% 37% 32 3% 40% 9% 37% 56% 0% 37% 38% 26% 39% 40% 0% 14% 0% 10% 31% 11% Ticks from Felines

Results: Rickettsia in Ticks Tick Pos/Tested (%) R. amblyommiiR. montanensisOther? A. 42/106 (40%) 5100 Aa 268/715 (37%) conorii 2 tarasevichiae 1 rickettsii 2 Am 65/166 (39%) 4303 parkeri1 Dv 10/212 (5%) 1100 Is 129/340 (38%) Rs 31/356 (9%) 3000 Total 545/1895 (29%)

Results: Rickettsia Incidence Ticks from Pets (n = 425/1580 ticks) Ticks from Deer (n = 120/315 ticks)

Results: Rickettsia Incidence Ticks from Pets (n = 425/1580 ticks) Ticks from Deer (n = 120/315 ticks)

Results: Rickettsia Incidence 2007 Cases (n=121): Benton, Cleburne, Crawford, Faulkner, Hot Spring, Pope, Pulaski, Van Buren, White (>4 cases)

Results: Rickettsia Risk Areas

Results: Co-infection Tick Rickettsia Pos/Tested (%) Borrelia Pos/Tested (%) CaninesFelinesDeer A. 42/106 (40%) 11/110 (10%) 5 5 (1 B.l. & R. a.) 0 Aa 268/715 (37%) 178/719 (25%) 56 (6 B.l. & R. a.) (1 B.l. & R. p.) 04 Am 65/166 (39%) 51/178 (29%) 80 9 (1 B. l. & R. p.) Dv 10/212 (5%) 20/213 (14%) 010 Is 129/340 (38%) 252/693 (36%) Rs 31/356 (9%) 91/362 (25%) 401 Total 545/1895 (29%) 613/2275 (27%) 92649

Results: Co-infection Borrelia Risk MapRickettsia Risk Map Map Algebra: Add the 2 Maps

Results: Co-infection

Summary BorreliaRickettsia

All Tick Species Positive All Hosts Positive Borrelia burgdorferi & Borrelia lonestari All Tick Species Positive R. amblyommii, R. montanensis, R. parkeri, R. rickettsii Summary BorreliaRickettsia

Future Direction Determine Population Structure of Aa & Is Conduct Field Work in Designated Risk Areas Identify the Pathogens in Humans Determine Symptoms & Pathogenesis Determine Vector Competency… Identify Environmental Variables with Risk Areas

Acknowledgements Arkansas Veterinary Medical Association University of Arkansas –Dr. D. Steelman –Dr. A. Szalanski –Dr. K. Loftin –Dr. K. Kvamme University of North Texas –Phillip Williamson –Peggy Billingsly –Janel Veterinary Entomology Laboratory –J. Shen Insect Genetics Laboratory –J. McKern –M. Tolivar Friends and Family